The Human Immunodeficiency Virus (generally referred to as HIV) continues to be a major problem worldwide, even though a plethora of compounds have been approved for treatment. Due to the error-prone nature of viral reverse transcriptase and the high viral turnover (t½=1-3 days), the HIV genome mutates very rapidly. For example, reverse transcriptase is estimated to generate, on average, one mutation per replication of the 9.7 Kb genome that does not dramatically affect the ability of the virus to propagate. This leads to the formation of ‘quasispecies’, where many different mutants exist in a dynamic relationship. HIV virus particles enter cells via the CD4 receptor and a co-receptor molecule. The co-receptor specificity of a given viral particle determines its tropism (The term “tropism” generally refers to the affinity of a viral particle for particular cell and receptor types). The majority of HIV-1 strains utilize the chemokine receptors CCR5 (R5 tropism), CXCR4 (X4 tropism) or both (R5X4 or dual tropism). Most newly infected individuals appear to have predominantly R5 tropic virus, and the SI phenotype has been associated with late-stage HIV infection and low CD4 cell counts as well as accelerated progression to AIDS. Additional examples of HIV tropism strains and their relationship to disease progression are described in Poveda et al., AIDS 2006, 20:1359-1367; Jensen et al., AIDS Rev 2003; 5:104-112; Jensen et al., Journal of Virology May 2006, p. 4698-4704; Jensen et al., Journal of Virology, December 2003, p. 13376-13388; and Nelson et al., Journal of Virology, November 1997, p. 8750-8758, each of which is hereby incorporated by reference herein in its entirety for all purposes.
There is currently only one FDA-approved drug that inhibits HIV entry by disrupting its interaction with a co-receptor, but more are in development. Maraviroc (also known as Selzentry, which is marketed by Pfizer Inc.) is a small molecule CCR5 inhibitor. Current recommendations published by the FDA state that each patient's HIV population be tested for tropism before Maraviroc is prescribed. This is due to the fact that clonal analysis of HIV quasispecies in patients that failed treatment during Maraviroc clinical trials revealed that small amounts of X4 tropic viruses were present before treatment initiation and it is thought that selection against CCR5 entry gave the X4 virus an advantage over the majority R5 strains. This mode of resistance development is analogous to the emergence of resistance to the ‘classical’ HIV drugs, i.e, protease and reverse transcriptase inhibitors, where a significant subset of HIV infected subjects carry pre-existing resistant strains prior to drug exposure—most likely due to primary infection by exposure to virus from treatment-experienced individuals. In addition to this pathway, resistant strains are continually generated de novo from wild type virus by replication under drug selective pressure due to the error-prone nature of the viral reverse transcriptase as described above. However, outgrowth of pre-existing resistant or, in the case of maraviroc treatment, X4 tropic virus, is more efficient under drug treatment and leads to accelerated treatment failure.
Viral tropism is determined by exposed amino acid sequences in the gp120 surface envelope protein. In particular, the V3 (third variable) region has been implicated in co-receptor usage selection. As the name implies, the approximately 35 amino acid long sequence is highly variable, but there are common features distinguishing R5 and X4 tropic viruses located within this sequence. A number of tropism prediction algorithms have been developed based directly on V3 sequences, and these are likely to be continually refined over the next few years. For example, several position specific scoring matrix (PSSM) algorithms that directly correlate amino acid residues in the V3 to tropism phenotypes (as well as these can be determined) have been published and some can even be accessed through the Internet.
Phenotypic tropism testing is now commercially available, but is expensive, labor intensive and time consuming. Additionally, phenotyping is heavily dependent on the efficient generation of a library of viral sequences, such that any cloning bias will generate a systematic testing error. Both phenotypic and sequence-based tropism determination is currently performed as population assays, which are, by their nature, less sensitive than assays based on clonal separation of each viral strain. However, clonal analysis is extremely labor intensive and requires testing of thousands of clones from each subject in order to achieve high sensitivity. Embodiments of the described invention include a sequence-based tropism determination assay wherein clonal sequences are obtained directly from viral RNA quasispecies without a labor intensive cloning step. Long read-length 454 sequencing available from 454 Life Science Corporation is ideally suited to generating thousands of clonal reads from multiple subjects in a single sequencing run. Further, embodiments of the described sequencing technologies enable what may be referred to as “Massively Parallel” capable of achieving a sensitivity of detection of low abundance variants that include a frequency of 1% or less of the allelic variants in a population. This, coupled with a tropism prediction algorithm provides a convenient method for quickly and efficiently obtaining tropism information at very high sensitivity.